Rifamycins P, Q and U

ABSTRACT

This invention is directed to rifamycin compounds which are produced by fermentation of Nocardia mediterranea ATCC 31064, ATCC 31065 or ATCC 31066 in an aqueous nutrient medium under aerobic conditions. These rifamycins are referred to as rifamycin P, rifamycin Q, rifamycin R and rifamycin U.

This invention is directed to novel rifamycin compounds which areproduced by fermentation of mutant strains of Streptomyces mediterraneiin an aqueous nutrient medium under aerobic conditions. These novelrifamycins are hereinafter referred to as rifamycin P, rifamycin Q,rifamycin R and rifamycin U.

It has previously been reported that during fermentation in normalgrowth media, Streptomyces mediterranei synthesizes a family ofantibiotics collectively referred to as the rifamycin complex (P. Sensi,et al., Antibiotics Annual 1959-1960, page 262). Subsequent workrevealed that the addition of sodium diethyl barbiturate to the culturemedium resulted essentially in the formation of a single fermentationproduct, rifamycin B (Margalith P. and Pagani H., Applied Microbiology,9, 325, 1961). According to a further discovery, a mutant strain ofStreptomyces mediterranei was found which elaborates essentiallyrifamycin B only, irrespective of the presence or absence of sodiumdiethyl barbiturate in the fermentation medium, so that an improvedprocess for making rifamycin B results. The rifamycin B producing strainwas identified as Streptomyces Mediterranei ATCC 21789. See U.S. Pat.No. 3,871,965.

The new antibiotic substances which are the object of this invention areproduced by mutant strains derived from Streptomyces mediterranei ATCC13685. The new strains are obtained by treating strain ATCC 13685 withcommon chemical mutagenic agents such as nitrous acid or nitrosoguanidine derivatives or with physical mutagenic agents such as X raysand U.V. radiation. The new mutant strains rather than to the genusStreptomyces mediterranei are assigned to the genus Nocardia inaccordance with the proposals by J. E. Thiemann et al., Arch.Mikrobiol., 67, 147-155 (1969).

Isolation of the mutated strains producing novel rifamycins

A suspension of spores of Streptomyces mediterranei ATCC 13685 wastreated with N-methyl-N'-nitro-N-nitrosoguanidine at 1 mg/ml in a pH 9.0tris(hydroxymethyl)aminomethane buffer for 60 minutes at 28° C. Themutagen-treated spores were then washed and plated onto Petri dishescontaining Bennett agar. After 14 days incubation at 28° C., thesurviving colonies were picked off and examined for their ability toinhibit the growth of Bacillus Subtilis in the following manner: a discof agar (diameter 4-8 mm) carrying a single colony was transferred ontoa Petri disch containing Penassay agar at pH 7.2 previously seeded at 2%(v/v) with Bacillus Subtilis.

Under these conditions rifamycin B is practically inactive and a normalcolony producing rifamycin B gives no inhibition of the growth ofBacillus subtilis in the underlying agar, whereas any colony producing anovel rifamycin with antimicrobial activity will cause a clear cut zoneof inhibition of growth around the disc of agar. Three strains wereisolated in this way and were originally given our internal code D-2,MM18-6 and M-36. Samples of these microorganisms were deposited with theATCC where they are identified respectively as Nocardia mediterraneaATCC 31064 Nocardia mediterranea ATCC 31065 and Nocardia mediterraneaATCC 31066.

Description of the new Nocardia Mediterranea strains

Table 1 reports the results of the macroscopic and mycroscopicexamination of the Nocardia mediterranea strains identified respectivelywith the ATCC No. 31064, 31065 and 31066, and Table 2 reports thecultural characteristics of the same Nocardia mediterranea strains.

The characteristics of the parent strain Streptomyces mediterranei ATCC13685 (ME/83) are also reported:

                                      TABLE 1                                     __________________________________________________________________________                  Colonies       Aerial mycelium                                                                            Spores                              __________________________________________________________________________    N. mediterranea                                                                           Colonies conical-crateriform and                                                               Greyish to pinkish,                                                                      Cylindrical                           ATCC 31064  convolute, 2-3 mm. of diameter,                                                                branched, 0.6-0.8                                                                        (0.8-1 μm × 3.0-5.0                                                  μ)                                             reddish-brown with ochre yellow                                                                μm of diameter                                (D-2)       soluble pigment                                                   N. mediterranea                                                                           Colonies conical-crateriform and                                                               Pink to light orange                                                                     Cylindrical                           ATCC 31065  convulate, 2-3 mm. of diameter,                                                                branched, 0.6-0.8                                                                        (0.8-1 μm × 3.0-5.0μ)                                             9                                                 deep orange with deep orange-                                                                  μm of diameter                                (MM18-6)    yellow soluble pigment                                            N. mediterranea                                                                           Colonies conical-crateriform and                                                               Pink to pink, orange                                                                     Cylindrical                           ATCC 31066  convolate, 2-3 mm. of diameter,                                                                branched, 0.6-0.8                                                                        (0.8-1 μm × 3.0-5.0μ)                                             2                                                 orange brown with amber soluble                                                                μm of diameter                                (M-36)      pigment                                                           Streptomyces medi-                                                                        Colonies conical-crateriform and                                                               Pinkish, branched,                                                                       Cylindrical                           terranei ATCC 13685                                                                       convolate, 2-3 mm of diameter,                                                                 0.6-0.8 μm of dia-                                                                    (0.8-1 μm × 3.0-5.0                                                  μ)                                             orange-yellow with light amber                                                                 meter                                            ME/83       soluble pigment                                                   __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________                                                     Streptomyces                          N. mediterranea                                                                            N. meditteranea                                                                            N. mediterranea                                                                             mediterranea                          ATCC 31064   ATCC 31065   ATCC 31066    ATCC 13685                   Media    (D-2)        (MM 18-6)    (M-36)        (ME/83)                      __________________________________________________________________________    Oat meal agar                                                                          Abundant growth with                                                                       Abundant growth with                                                                       Abundant with smooth                                                                        Fair growth with smooth               slightly wrinkled surfa-                                                                   smooth surface, orange.                                                                    face, deep amber.                                                                           surface, yellowish with               ce, hazel-brown with                                                                       Traces of pinkish aerial                                                                   Traces of pink aerial                                                                       pinkish reverse.                                                              Whitish                               orange edges. Pink-beige                                                                   mycelium. Some spores.                                                                     mycelium. Burnt amber                                                                       aerial mycelium with                                                          pink                                  soluble pigment.                                                                           Pink-beige soluble pig-                                                                    soluble pigment.                                                                            tinge. Traces of                                                              yellow-                                            ment.                      ish soluble pigment.         Yeast extract                                                                          Abundant growth with                                                                       Abundant growth with                                                                       Abundant growth with                                                                        Abundant growth with         glucose agar                                                                           very wrinkled surface,                                                                     very wrinkled surface,                                                                     wrinkled surface. Vege-                                                                     rough surface yellowish      (Medium No. 2                                                                          amber. Greyish aerial                                                                      brick red. Deep rusty                                                                      tative mycelium, burnt                                                                      to pink. Scanty aerial       Shirling and                                                                           mycelium, deep rusty                                                                       soluble pigment.                                                                           amber. Burnt amber so-                                                                      mycelium.                    Gottlieb)                                                                              soluble pigment.          luble igment.                              Emerson's glu-                                                                         Abundant growth with                                                                       Abundant growth with                                                                       Abundant growth with                                                                        Abundant growth with         cose agar                                                                              very wrinkled and cru-                                                                     very wrinkled and cru-                                                                     wrinkled surface, burnt                                                                     rough surface,                                                                yellowish                             sty surface, brick red                                                                     sty surface, brick red.                                                                    amber. Burnt amber solu-                                                                    to pink orange. Pinkish               Amber soluble pigment.                                                                     Amber soluble pigment.                                                                     ble pigment.  aerial mycelium. Pale                                                         amber soluble pigment.       Bennett's                                                                              Abundant growth, very                                                                      Abundant growth with                                                                       Abundant growth with                                                                        Good growth, yellowish       agar     wrinkled, reddish brown                                                                    very wrinkled surface,                                                                     wrinkled surface, orange                                                                    turning orange-yellow.                Traces of greyish aerial                                                                   deep orange. Deep yellow                                                                   brown. Amber soluble                                                                        Pinkish aerial                                                                mycelium.                             mycelium. Ochre yellow                                                                     soluble pigment.                                                                           pigment.      Light amber soluble                                                           pigm-                                 soluble pigment.                        ent.                         Penassay agar                                                                          Moderate growth, sligh-                                                                    Moderate growth, sligh-                                                                    Moderate growth with                                                                        Poor growth                           tly wrinkled light                                                                         tly wrinkled light oran-                                                                   smooth and thin surface,                            orange. No soluble pig-                                                                    ge. No soluble pigment.                                                                    light orange. No soluble                            ment.                     pigment.                                   Yeast extract                                                                          Abundant growth                                                                            Abundant growth with                                                                       Abundant growth with                                                                        Abundant growth with                                                          rough                        molasse agar                                                                           very wrinkled, brick red                                                                   very wrinkled surface,                                                                     sty surface, burnt amber                                                                    surface, colorless to                 Deep rusty soluble pig-                                                                    brick red. Deep rusty                                                                      Burnt amber soluble pig-                                                                    yellowish. Whitish                                                            aerial                                ment.        soluble pigment.                                                                           ment.         mycelium. Deep amber                                                          solu-                                                                         ble pigment.                 Czapek-Dox                                                                             Moderate growth with                                                                       Good growth with smooth                                                                    Abundant growth with                                                                        Poor growth with thin        saccharose                                                                             smooth and thin surface                                                                    surface, orange. Light                                                                     smooth surface, salmon.                                                                     surface, colorless to        agar     light orange. Pale pink                                                                    orange aerial mycelium                                                                     Abundant aerial mycelium                                                                    light melon. Traces of                aerial mycelium. Some     pink orange. Traces of                                                                      pinkish white aerial                  spores. Deep lemon yellow light yellow soluble                                                                        mycelium                              soluble pigment.          pigment.                                   Potato agar                                                                            Scarce growth, light oran-                                                                 No growth    Moderate growth with                                                                        Poor growth with thin                 ge. Light hazel brown     smooth surface. Light                                                                       surface, colorless.                   soluble pigment.          orange. No soluble pig-                                                                     Traces of whitish                                                             aerial                                                          ment.         mycelium. No soluble                                                          pigment.                     Glucose  Abundant growth with                                                                       Scarce growth with smooth                                                                  Fair growth, gold.                                                                          Fair growth with smooth      asparagine                                                                             smooth surface orange.                                                                     surface, light orange.                                                                     No soluble pigment.                                                                         and thin surface, light      agar     Light yellow soluble                                                                       No soluble pigment.        orange pink. Some light               pigment.                                yellow soluble pigment.      Glycerol Abundant growth with                                                                       Scarce growth, deep                                                                        Moderate growth with                                                                        Fair growth with smooth      asparagine                                                                             smooth surface deep                                                                        orange.      slightly wrinkled surfa-                                                                    and thin surface. Light      agar (medium                                                                           orange. Traces of                                                                          No soluble pigment.                                                                        ce, deep orange. Traces                                                                     orange pink. Some light      n° 5 Shirling                                                                   yellowish soluble pig-    of yellow soluble pig-                                                                      yellow soluble pigment.      and Gottlieb)                                                                          ment.                     ment.                                      Nutrient agar                                                                          Moderate growth with                                                                       Moderate growth with                                                                       Fair growth with thin                                                                       Moderate growth with                                                          smooth                                thin and smooth surfa-                                                                     wrinkled and thin sur-                                                                     and smooth surface,                                                                         surface, melon to                                                             orange.                               ce, pale orange.                                                                           face, pale orange.                                                                         orange. No soluble                                                                          Pinkish white aerial                                                          my-                                   No soluble pigment.                                                                        No soluble pigment.                                                                        pigment.      celium.                      Pridham's                                                                              Moderate growth with                                                                       Abundant growth with                                                                       Abundant growth, sligh-                                                                     Moderate growth with         agar     smooth and thin surface,                                                                   smooth surface, brick                                                                      tly crusty, amber brown.                                                                    smooth surface,                                                               colorless                             amber. Light pinkish-                                                                      red. Pink aerial myce-                                                                     Traces of pinkish aerial                                                                    with lobster red spots.               orange aerial mycelium.                                                                    lium. Yellow soluble                                                                       mycelium.     Pink aerial mycelium.                 Pinkish soluble pigment.                                                                   pigment.     Amber brown soluble pig-                                                      ment.                                      Starch agar                                                                            Moderate growth with                                                                       Moderate growth with                                                                       Abundant growth with                                                                        Poor growth, colorless                                                        to                           (medium n° 4                                                                    smooth surface orange.                                                                     smooth surface, deep                                                                       smooth surface, orange.                                                                     light orange pink.           Shirling and                                                                           Abundant aerial myce-                                                                      orange.      Traces of pinkish aerial                                                                    Scarce white aerial                                                           myce-                        Gottlieb)                                                                              lium, pink. Abundant                                                                       Traces of straw soluble                                                                    mycelium. Light yellow                                                                      lium. Starch                                                                  hydrolysis:                           production of spores.                                                                      pigment. Starch hydro-                                                                     soluble pigment. Starch                                                                     doubtful                              Yellow soluble pigment.                                                                    lysis: negative.                                                                           hydrolysis: negative.                               Starch hydrolysis: nega-                                                      tive.                                                                Dextrose trip-                                                                         Abundant growth with                                                                       Moderate growth, wrin-                                                                     Abundant growth with                                                                        Abundant growth pink         tone agar                                                                              wrinkled surface melon.                                                                    kled, orange. Orange                                                                       wrinkled surface, rose-                                                                     orange. Pinkish aerial                Orange melon soluble                                                                       yellow soluble pigment.                                                                    melon. Rose melon solu-                                                                     mycelium. Light golden                pigment.                  ble pigment.  yellow soluble pigment.      Hickey's and                                                                           Moderate growth with                                                                       Abundant growth with                                                                       Moderate growth with                                                                        Moderate growth, light       Tresner's                                                                              smooth and thin surface,                                                                   smooth surface, rose                                                                       slightly wrinkled surfa-                                                                    pinkish orange. Some         cobalt agar                                                                            colorless. Whitish aerial                                                                  melon. Light beige-rose                                                                    ce, rose-melon. Light                                                                       pinkish aerial                                                                mycelium.                             mycelium. Beige soluble                                                                    soluble pigment.                                                                           beige soluble pigment.                                                                      Yellowish soluble                                                             pigment.                              pigment.                                                             Tyrosine agar                                                                          Abundant growth, very                                                                      Abundant growth, very                                                                      Abundant growth with                                                                        Poor growth                  (Medium n° 7                                                                    wrinkled and crusty                                                                        wrinkled and crusty.                                                                       crusty surface, deep                       Shirling and                                                                           brick red. Deep yellow                                                                     Deep orange. Pinkish-                                                                      burnt orange. Yellow                       Gottlieb)                                                                              soluble pigment. Tyrosi-                                                                   tan soluble pigment.                                                                       ochre soluble pigment.                              ne reaction: positive                                                                      Tyrosine reaction:                                                                         Tyrosine reaction:                                  (good).      strongly positive.                                                                         strongly positive.                         Ca-malate                                                                              Scarce growth with                                                                         Scarce growth, light                                                                       Poor growth, light                                                                          Fair growth colorless,       agar     smooth and thin surface,                                                                   orange. Hydrolysis of                                                                      orange. No soluble pig-                                                                     Whitish pink aerial                   light orange yellow.                                                                       Ca-malate: negative.                                                                       ment. Ca-malate dige-                                                                       mycelium. Partial dige-               Chrome lemon soluble      stion: negative.                                                                            stion of Ca-malate.                   pigment near the growth                                                       and coral pink in the                                                         medium. Hydrolysis: po-                                                       sitive.                                                              Egg albumin                                                                            Abundant growth with                                                                       Abundant growth with                                                                       Good growth with smooth                                                                     Fair growth, pink.           agar     smooth surface, light                                                                      smooth surface, orange.                                                                    surface orange-yellow.                              orange. Light yellow                                                                       No soluble pigment.                                                                        Light yellow soluble pig-                           soluble pigment.          ment.                                      Peptone glu-                                                                           Abundant growth, sligh-                                                                    Abundant growth, wrin-                                                                     Abundant growth with                       cose agar                                                                              tly wrinkled, deep oran-                                                                   kled, deep orange.                                                                         wrinkled surface, burnt                             ge. Pinkish aerial myce-                                                                   Gold yellow soluble                                                                        amber. Burnt amber so-                              lium. Yellow soluble pig-                                                                  pigment.     luble pigment.                                      ment.                                                                Gelatine hydrolysis: positive                                                                       hydrolysis: positive                                                                       hydrolysis: positive                                                                        hydrolysis: positive         Nitrate broth                                                                          reduction: negative                                                                        reduction: negative                                                                        reduction: negative                                                                         reduction: negative          Litmus milk                                                                            no coagulation                                                                             no coagulation                                                                             no coagulation                                                                              no coagulation                        no peptonization                                                                           no peptonization                                                                           no peptonization                                                                            no peptonization             Peptone yeast                                                                          Moderate growth with                                                                       Moderate growth with                                                                       Poor growth with                           extract iron                                                                           smooth surface, pale                                                                       smooth surface, pale                                                                       smooth surface, color-                     agar (Medium                                                                           orange. H.sub.2 S production:                                                              orange. H.sub.2 S production:                                                              less. No soluble pig-                      n° 6 Shirling                                                                   negative     negative.    ment. H.sub.2 S production:                and Gottlieb)                      negative.                                  Potato plug                                                                            Scarce growth, orange.                                                                     Very scarce growth,                                                                        Poor growth, light o-                                                                       Poor growth, colorless.                            orange.      range.                                     Loeffler No growth    No growth    No growth                                  blood serum                                                                   H.sub.2 O+agar                                                                         Scarce growth colorless.                                                                   Scarce growth, colorless.                                                                  Scarce growth, colorless                   Difco 2% Traces of white aerial                                                                     Traces of white aerial                                                                     Traces of white aerial                              mycelium. Abundant pro-                                                                    mycelium. Abundant pro-                                                                    mycelium. Abundant pro-                             duction of spores.                                                                         duction of spores.                                                                         duction of spores.                         Skim milk agar                                                                         Abundant growth with                                                                       Good growth, slightly                                                                      Abundant growth with                                smooth surface, orange.                                                                    wrinkled, orange with                                                                      wrinkled surface, oran-                             Cadmium yellow soluble                                                                     brown shadow.                                                                              ge. Gold brown soluble                              pigment. Hydrolysis of                                                                     Hazel brown soluble                                                                        pigment. casein hydroly-                            casein: good.                                                                              pigment. Hydrolysis of                                                                     sis: strongly positive.                                          casein: good.                                           __________________________________________________________________________     Shirling and Gottleib: Methods for characterization of Streptomyces           species, Intern. J. Syst. Bact., 16, 313-338 (1966).                     

                                      TABLE 3                                     __________________________________________________________________________            N. mediterranea                                                                        N. mediterranea                                                                        N. mediterranea                                                                         Streptomyces mediterranei                         (D-2)    (MM 18-6)                                                                              (M-36)    (ME/83)                                   __________________________________________________________________________    Strach hydro-                                                                 lysis    -       -        -        ±                                       Tyrosin hydro-                                                                lysis   ++       +++      +++      -                                          Casein hydro-                                                                 lysis   ++        ++      +++                                                 Ca-malate                                                                     hydrolysis                                                                            ++       -        -        +                                          Nitrate re-                                                                   duction  -       -        -        -                                          Litmus milk                                                                           no coagulation                                                                         no coagulation                                                                         no coagulation                                                                         no coagulation                                     no peptonization                                                                       no peptonization                                                                       no peptonization                                                                       no peptonization                           Gelatin                                                                       liquefaction                                                                          ++        ++       ++      +                                          __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________           N. mediterranea                                                                        N. mediterranea                                                                        N. mediterranea                                                                        Streptomyces mediterranei                          (D-2)    (MM 18-6)                                                                              (M-36)   (ME/83)                                     __________________________________________________________________________    Arabinose                                                                              +        +++      ++       +++                                       Xylose   +        +        ++       ++                                        Glucose  ++       +++      ++       ++                                        Mannose  +        ++       ++       ++                                        Fructose ++       +++      ++       ++                                        Lactose  +        +++      ++       ++                                        Sucrose  +        ±     ++       ++                                        Inositol +        +        ++       ++                                        Rhamnose +        +        ++       ++                                        Raffinose                                                                              -        -        -        -                                         Salicin  ±     +        +        +                                         Mannitol ++       +++      ++       ++                                        Glicine  +        ±     +        -                                         Sorbitol -        -        -        -                                         Ma-succinate                                                                           -        -        -        +                                         Na-citrate                                                                             -        -        -        -                                         Na-acetate                                                                             -        -        -        -                                         Inulin   ±     +        -        -                                         Dulcitol -        -        -        -                                         Cellulose                                                                              -        -        -        -                                         Maltose  ++       +++      ++       +                                         Galactose                                                                              +        ++       ++       +++                                       Glicerol ++       +++      ++       +++                                       __________________________________________________________________________

FERMENTATION

The procedure for the fermentation essentially consists in cultivatingone of said mutants of Streptomyces mediterranei in a nutrient mediumcontaining assimilable carbon and nitrogen sources and essential mineralsalts, until a substantial antibiotic activity is imparted to saidmedium and in extracting the rifamycins from the medium. Moreparticularly these mutants are cultivated under stirred and aeratedsubmerged conditions at a temperature ranging from 25° to 37° C., andpreferably at 28° C. As the sources of carbon the followingcarbohydrates and carbon derivatives can be used: glucose, galactose,lactose, sucrose, maltose, glycerol, mannitol, etc. Useful nitrogensources are for instance amino-acids and their mixtures, peptides,proteins and their hydrolisates, as peptone, yeast extract, soybeanmeal, corn steep liquor, fish soluble, meet extracts, aqueous fractionsfrom cereal seeds. The fermentation can be carried out for 180-220hours. The starting pH, generally adjusted at about 6.4-6.6 increases atthe end of the fermentation to 7.0-8.5. Generally the best results areobserved at the 200 hours of the fermentation. At the end of thefermentation the rifamycins can be isolated by the following procedure.The fermentation medium is filtered at the final pH of 7.0-8.5. Thefiltrate is quickly acidified, preferably to a pH lower than about 5, toensure the best stability to the antibiotic substance. The activity isextracted with water immiscible solvents such as chloroform, butanol,ethyl, propyl, butyl or amyl acetate. The ratio between the volume ofthe medium and that of the solvent changes dependently on the choosensolvent: generally a ratio ranging from 2:1 to 10:1 is used.

The mycelium still retains a microbiological activity, which isextracted from the mycelium by means of a water immiscible solvent, andthen combined with the organic phase, already containing most of therifamycins. Alternatively, the extraction of the activity from themycelium can be effected by means of a solvent miscible with water suchas acetone. In this case, the liquid is filtered, the acetone isevaporated in vacuo, the rifamycins are extracted with a waterimmiscible solvent and the procedure is carried on as said above.

Once most of the antibiotic activity has been transferred into thesolvent, this is distilled in vacuo to dryness, preferably at atemperature lower than 30° C.

PURIFICATION

The crude extract of rifamycins can be purified chromatographically on acolumn of silica-gel. Prior to chromatography it is convenient todissolve the crude extract in a phosphate buffer pH 7.0-8.0 and to treatwith a mild oxidizing agent. The buffer solution is then extracted witha water-immiscible solvent, this organic extract contains a newrifamycin denominated rifamycin R. The extracted buffer solution isacidified to pH 2-4 and then again extracted with a water immisciblesolvent. This organic extract contains three new compounds of therifamycin family denominated rifamycin P, Q and U.

The further purification of rifamycin R is effected by chromatography ona column of suitable adsorbing material such as silica-gel and elutingwith an appropriate mixture of organic solvents.

The second organic extract containing rifamycins P, Q and U is purifiedin a similar way to that described for rifamycin R.

The following examples are given for the purpose of better illustratingthe subject of the invention.

EXAMPLE 1

The mutant strains identified as Nocardia mediterranea ATCC 31064 ispropagated for 6-8 days on Bennett's agar and incubated at 28° C.

With the culture obtained from the agar slant, two 500 ml. Erlenmeyerflasks are inoculated under sterile conditions. The flasks contain 100ml. of the vegetative medium of the following composition:

    ______________________________________                                        Beef extract      5        g.                                                 Yeast extract     5        g.                                                 Peptone           5        g.                                                 Casein hydrolyzate                                                                              3        g.                                                 Glucose           20       g.                                                 NaCl              1.5      g.                                                 H.sub.2 O to      1        liter.                                             ______________________________________                                    

The pH is adjusted to 7.3 with NaOH.

The flasks so inoculated are placed on an alternative shaker at 28° C.for 72 hours. The content of the two Erlenmeyer flasks is used asinoculum by pouring it in a 10 liters prefermenter, containing 4 litersof the above mentioned vegetative medium. The incubation is carried outat 28° C. with an agitation of 300 r.p.m. and 1 v/v/m aeration. After 48hours of growth a volume of 7-10% of packed cells is obtained. In thenext stage a 10 liter glass fermenter containing 4 liters of thehereinafter mentioned fermentation medium is used:

    ______________________________________                                        Peanut flour           25       g.                                            Soybean flour          5        g.                                            (NH.sub.4).sub.2 SO.sub.4                                                                            9.5      g.                                            NgSO.sub.4. 7H.sub.2 O 0.85     g.                                            Glucose                95       g.                                            Glycerol               40       g.                                            KH.sub.2 PO.sub.4      1        g.                                            Propylene glycol       5        g.                                            CaCO.sub.3             8.5      g.                                            Na diethylbarbiturate  1.7      g.                                            CuSO.sub.4 . 5H.sub.2 O                                                                              2.8      mg.                                           FeSO.sub.4 . 7H.sub.2 O                                                                              8.5      mg.                                           ZnSO.sub.4 . 7H.sub.2 O                                                                              42.5     mg.                                           MnSO.sub.4 . 4H.sub.2 O                                                                              3.4      mg.                                           CaCl.sub.2 . 6H.sub.2 O                                                                              1.7      mg.                                           (NH.sub.4).sub.6 Mo.sub.7 O.sub.24 . 4H.sub.2 O                                                      0.85     mg.                                           H.sub.2 O to           1        liter                                         ______________________________________                                    

The pH is adjusted to 7.8 with NaOH. Sterilization for 60 minutes at120° C. After sterilization the pH is 6.4. An amount of the prefermentercontent equal to 5% of the fermenter content is used as inoculum.

The fermentation is carried out at 28° C. with a 750 r.p.m. agitationand aerating at a rate of 1 v/v/m. Silicone A is used as antifoam. Theculture broth turns to a characteristic red-brown color during thefermentation. After 200 hours of growth a volume of packed cells isobtained. The pH of the broth is 7.5 and at this moment the broth isharvested.

EXAMPLE 2

A culture of Nocardia mediterranea ATCC 31064 obtained as disclosed inexample 1, is prepared in a flask, under stirring, as described inexample 1. For the preculture it is poured into a 10 liters glassfermenter, containing 4 liters of the following medium:

    ______________________________________                                        Glucose             5        g.                                               Peanut flour        7.5      g.                                               CaCO.sub.3          1.65     g.                                               MgSO.sub.4 . 7H.sub.2 O                                                                           0.33     g.                                               KH.sub.2 PO.sub.4   0.33     g.                                               FeSO.sub.4 . 7H.sub.2 O                                                                           3.3      mg.                                              ZnSO.sub.4 . 7H.sub.2 O                                                                           16.5     mg.                                              MnSo.sub.4 . 4H.sub.2 O                                                                           1.3      mg.                                              H.sub.2 O to        1        liter                                            ______________________________________                                    

The pH is adjusted to 7.5. Sterilization 50 minutes at 120° C. Aftersterilization the pH value is 6.4. After 48 hours of growth the volumeof packed cells is 6-8% of total volume. An inoculum equal to 10% isused for a 20 liters glass fermenter, containing 10 liters of thefollowing fermentation medium:

    ______________________________________                                        Corn steep liquor  20        g.                                               Soybean flour      15        g.                                               (NH.sub.4).sub.2 SO.sub.4                                                                        6         g.                                               MgSO.sub.4 . 7H.sub.2 O                                                                          0.85      g.                                               Glucose            100       g.                                               KH.sub.2 PO.sub.4  1         g.                                               CaCO.sub.4         6         g.                                               FeSO.sub.4 . 7H.sub.2 O                                                                          8.5       mg.                                              ZnSO.sub.4 . 7H.sub.2 O                                                                          42.5      mg.                                              MnSO.sub.4 . 4H.sub.2 O                                                                          3.4       mg.                                              CuSO.sub.4 . 5H.sub.2 O                                                                          2.8       mg.                                              CoCl.sub.2 . 6H.sub.2 O                                                                          1.7       mg.                                              H.sub.2 O to       1         liter                                            ______________________________________                                    

The pH is adjusted to 7.8 with NaOH. Sterilization for 50 minutes at120° C. After sterilization the pH is 6.4. The fermentation is carriedout at 28° C. for 200 hours. The pH of the fermentation broth at theharvest is 7.5.

The fermentation broths obtained in examples 1 and 2 are purified in thefollowing way. The mycelium is removed by filtration and discarded, thefiltrate is adjusted to pH 2.0 with 10% (v/v) hydrochloric acid andextracted three times with an equal volume of ethyl acetate. Thisorganic extract is concentrated to dryness under vacuum at 35° C. andthe residue (4 g. from example 1 and 11 g. from example 2) is dissolvedin 0.05 M sodium phosphate buffer pH 7.5 and sodium nitrite added togive a final concentration of 0.2% (w/v). After stirring for 30 min. atroom temperature the buffer solution is extracted three times with anequal volume of ethyl acetate. The combined organic extracts areconcentrated to dryness under vacuum at 35° C. (1^(st) ethyl acetateextract). The exhausted buffer solution is adjusted to pH 2.0 with 10%hydrochloric acid and extracted with an equal volume of ethyl acetatethree times. The combined organic extracts are concentrated to drynessunder vacuum at 35° C. (2^(nd) ethyl acetate extract).

The dry powder from the 1^(st) ethyl acetate extract (1.4 g example 1and 4.2 g. example 2) is dissolved in chloroform and chromatographed ona column of silica-gel (70-230 Mesh ASTM) using chloroform containing 2%(v/v) methyl alcohol as an eluent. Rifamycin R is the first majorproduct to elute from the column recognizable by its orange-browncolour.

On thin layer chromatography using silica-gel plates (Merck 60 F₂₅₄)with chloroform: methyl alcohol as solvent system (95:5) rifamycin R hasan Rf of 0.59. The fractions containing rifamycin R are combined, takento dryness under vacuum at 35° C., redissolved in ethyl acetate which isthen washed with 0.01 N hydrochloric acid and finally with water. Theorganic extract is concentrated to a small volume and rifamycin Rcrystallizes from solution at 4° C. (obtained 600 mg. from example 1 and1.6 g. from example 2). The second ethyl acetate extract is purified bycolumn chromatography on silica-gel in a similar way to rifamycin R. Thedry residue (2.4 g. from example 1 and 4.2 g. from example 2) isdissolved in chloroform and applied to the silica gel.

The column is eluted with a mixture of chloroform and methyl alcohol(98:2). The first compound to emerge is rifamycin U, then rifamycin Pand finally rifamycin Q. All three of these compounds have a yelloworange colour in the eluent solution and can be identified on the basesof their mobility on thin layer chromatography. On silica gel plates(Merck 60 F₂₅₄) using chloroform:methanol (95:5) as solvent system, theRf values are the following:

    ______________________________________                                        rifamycin U          Rf = 0.63                                                rifamycin P          Rf = 0.57                                                rifamycin Q          Rf = 0.32                                                ______________________________________                                    

The appropriate fractions containing rifamycins U, P and Q are combinedtaken to dryness under vacuum and crystallized from ethyl acetate. Fromthe first example are obtained rifamycin U 80 mg., rifamycin P 400 mg.,rifamycin Q 280 mg: from the second example rifamycin U 190 mg.,rifamycin P 900 mg., rifamycin Q 750 mg.

By following essentially the same procedure of the above examples butusing respectively Nocardia mediterranea ATCC 31065 or Nocardiamediterranea ATCC 31066 as the producing strains, yields of the sameorder as above are obtained.

Physico-chemical characteristics of the novel rifamycins Rifamycin P

1. Elemental analysis (%): Found: C=60.6; H=6.2; N=3.8; O=25.2; S=4.1

2. U.V. and visible absorption bands

The compound shows the following values:

    ______________________________________                                        methanol               0.1 N HCl                                              ______________________________________                                        λ.sub.max(mμ)                                                                 E.sub.1 cm.sup.1%                                                                          λ.sub.max(mμ)                                                                  E.sub.1 cm.sup.1%                           406       197          416        183                                         350       (shoulder)   300        319                                         297       344          225        521                                         257       423                                                                 224       550                                                                 ______________________________________                                    

The complete figure of the spectrum is given in FIG. 1. The spectrum wasrecorded with Perkin Elmer Spectracord 4000 A instrument.

3. Infrared Spectrum

The most significant absorption peaks in Nujol occur at the followingfrequencies (cm⁻ ¹):

3700-3150 (m,br); 3100 (w); 3060-2800 (vs); 1465 (s); 1380 (b): Nujol;1722 (m); 1645 (m,br); 1580 (m); 1510 (m); 1325 (m); 1250 (s br); 1160(m); 1130 (w); 1070 (m, br) 1030 (w); 982 (m); 960 (m); 925 (w); 890(m); 818 (w); 770 (w); 735 (w).

A complete figure of the I.R. spectrum is given in FIG. 2. The spectrumwas recorded with a Perkin Elmer Mod. 421 instrument.

4. Mass spectrum

The mass spectrum obtained at 70 e V shows the molecular ion peak M⁺ atthe following m/e value: 738. The spectrum was recorded with an HitachiPerkin Elmer RMU-6L instrument.

5. Nuclear Magnetic Resonance spectrum

The complete figure of N.M.R. spectrum at 100 MH_(z) in CDCl₃ is givenin FIG. 3.

This compound does not show the characteristic polarographic behavior ofthe rifamycins possessing a chromophoric moiety with quinonic structure.

Rifamycin Q

1. Elemental analysis: Found: C=60.7; H=6.3; N=3.60; O=25.3; S=4.2

2. U.V. and visible absorption bands

The compound shows the following values in methanol:

    ______________________________________                                               λ.sub.max (mμ )                                                                 E.sub.1 cm.sup.1%                                          ______________________________________                                               406         178                                                               350         (shoulder)                                                        297         305                                                               257         405                                                               224         518                                                        ______________________________________                                    

3. Infrared Spectrum

The most significant absorption peaks in Nujol occur at the followingfrequencies (cm⁻ ¹):

3700-3300 (s,br); 3300-3080 (m br); 3040-2780 (vs); 1460 (s); 1378 (s):Nujol; 1740 (m); 1700 (m); 1650 (s,br); 1605 (s,br); 1555 (s); 1510(m,br); 1315 (w); 1275 (m); 1240 (m); 1220 (m); 1160 (m); 1090 (m); 1050(m,br); 1020 (w); 970 (m); 945 (w); 910 (m); 808 (m); 765 (w); 720 (w).

A complete figure of the I.R. spectrum is given in FIG. 4.

4. Nuclear Magnetic Resonance Spectrum

The complete figure of the N.M.R. spectrum at 60 MH_(z) in CDCl₃ isgiven in FIG. 7.

Rifamycin R

1. Elemental analysis: Found: C=62.0; H=6.4; N=2.2; O=29.6.

2. U.V. and visible absorption bands

The compound shows the following values in 0.1 N HCl in methanol:

    ______________________________________                                               λ.sub.max (mμ)                                                                  E.sub.1 cm.sup.1%                                          ______________________________________                                               219         444                                                               281         415                                                               340         114                                                               410         72                                                         ______________________________________                                    

3. Infrared Spectrum

The most significant absorption peaks in Nujol occur at the followingfrequencies (cm⁻ ¹):

3700-3100 (s br); 3040-2780 (vs); 1465 (s); 1380 (s): Nujol; 1745 (s);1710 (m); 1640 (s); 1600 (s); 1505 (s); 1415 (m); 1325 (s); 1260 (s,br);1220-1130 (s,br); 1120 (w); 1075 (s); 1020 (w); 975 (s); 950 (w); 920(w); 888 (m); 823 (m); 785 (w,br); 735 (w,br); 650 (w,br).

A complete figure of the I.R. spectrum is given in FIG. 5.

4. Mass spectrum

The mass spectrum obtained at 70 e V shows the molecular ion peak M⁺ atthe following m/e value: 711.

5. Nuclear Magnetic Resonance spectrum

The complete figure of the N.M.R. spectrum at 60 MH₂ in CDCl₃ is givenin FIG. 6.

The compound shows the characteristic polorographic behavior of therifamycins possessing a chromophoric moiety with quinonic structure.

The above data support the following structure for rifamycin R. ##STR1##

Rifamycin U

1. U.V. and visible absorption bands

The compound shows the following values in methanol:

    ______________________________________                                               λ.sup.max (mμ)                                                                  E.sub.1 cm.sup.1%                                          ______________________________________                                               412         163                                                               353         (shoulder)                                                        299         265                                                               258         389                                                               220         451                                                        ______________________________________                                    

2. Infrared spectrum

The most significant absorption peaks in Nujol occur the followingfrequencies (cm⁻ ¹):

3700-3100 (m,br); 3060 (vw); 3040-2740 (vs), 2720 (vw), 1460 (s), 1378(m): Nujol; 1750-1705 (m,br); 1680-1620 (m,br); 1600 (s); 1558 (s); 1490w,shoulder); 1308 (w); 1270 (w,shoulder); 1245 (m, shoulder); 1225 (m);1160 (m); 1120 (w); 1090 (vw); 1070 (w); 1030 (vw); 1020 (vw); 1000(vw); 975 (w); 960 (w, shoulder); 905 (m); 850 (m); 802 (w); 800 (vw,shoulder); 777 (vw); 760 (vw); 720 (vw); 685 (vw); 670 (vw).

BIOLOGICAL ACTIVITY OF THE COMPOUNDS

The in vitro activity spectrum of rifamycins P, Q and R is reported inthe following table:

    __________________________________________________________________________                       Minimal inhibiton concentration (μg/ml)                                    Rifamycin P                                                                           Rifamycin Q                                                                           Rifamycin R                                                                           Rifamycin U                        __________________________________________________________________________    Staphylococcus aureus ATCC 6538                                                                  0.00235 0.039   0.019   0.05-0.1                           Staphylococcus aureus ATCC 6538                                                                  0.00235 0.078   0.0047  --                                 (20% bovine serum)                                                            Staphylococcus aureus Tour                                                                       0.0035  0.06    0.00235 --                                 Staphylococcus aureus Tour                                                    rifampicin-resistant                                                                             25-50   >100    100     --                                 Streptococcus hemolyticus C 203                                                                  0.0047  0.019   0.00312 --                                 Streptococcus faecalis ATCC 10541                                                                0.0035  0.312   0.19    --                                 Diplococcus pneumoniae UC 41                                                                     0.0047  0.039   0.00625 --                                 Proteus vulgaris X 19 H ATCC 881                                                                 0.78    12.5    12.5    --                                 Escherichia coli ATCC 10536                                                                      3.12    12.5    50      >50                                Klebsiella pneumoniae ATCC 10031                                                                 12.5    50      --      --                                 Pseudomonas aeruginosa ATCC 10145                                                                12.5    50      100     --                                 Mycobacterium tub. H37Rv ATCC 9360                                                               1       2.5     0.1     2.5                                __________________________________________________________________________     --: Activity not determined                                              

The novel compounds are active also in the laboratory animals injectedwith Staphylococcus aureus, Escherichia coli. The following tablereports the results of representative experiments on mice in comparisonwith a known natural rifamycin, i.e. rifamycin SV.

    ______________________________________                                                              ED.sub.50 mg/kg.                                        Compound   Infecting Strain s.c.    o.s.                                      ______________________________________                                        rifamycin P                                                                              Staphylococcus aureus                                                                          0.3     0.8                                       rifamycin P                                                                              Escherichia coli 40      --                                        rifamycin Q                                                                              Staphylococcus aureus                                                                           8      --                                        rifamycin SV                                                                             Staphylococcus aureus                                                                          17      74                                        ______________________________________                                    

Rifamycin P which results to be the most active natural rifamycin so farisolated has also low toxicity since its LD₅₀ value in mice is higherthan 500 mg/kg. i.p.

We claim:
 1. A rifamycin compound defined as rifamycin P having the following characteristics:1. Elemental analysis (%): C=60.6; H=6.2; N=3.8; O=25.2; S=4.1
 2. u.v. and visible absorption bands (FIG. 1):

    ______________________________________                                         methanol               0.1 N HCl                                               ______________________________________                                         λ.sub.max(mμ)                                                                  E.sub.1 cm.sup.1%                                                                           λ.sub.max(mμ)                                                                   E.sub.1 cm.sup.1%                            406       197          416        183                                          350       (shoulder)   300        319                                          297       344          225        521                                          257       423                                                                  224       550                                                                  ______________________________________                                    


3. The most significant infrared absorptiion peaks in Nujol at the following frequencies (cm.⁻ ¹) (FIG. 2): 3700-3150 (m, br); 3100 (w); 3060-2800 (vs); 1465 (s); 1380 (b): Nujol; 1722 (m); 1645 (m, br); 1580 (m); 1510 (m); 1325 (m); 1250 (s br); 1160 (m); 1130 (w); 1070 (m, br) 1030 (w); 982 (m); 960 (m); 925 (w); 890 (m); 818 (w); 770 (w); 735 (w),
 4. A mass spectrum at 70 ev showing the molecular ion peak M⁺ at the following m/e value: 738,
 5. A complete figure of N.M.R. spectrum at 100 MH_(z) in CDCl₃ as in FIG. 3,
 6. rf value of 0.57 on silicagel plate using chloroform:methanol (95:5) as a solvent system.
 2. A rifamycin compound defined as rifamycin Q having the following characteristics:1. Elemental analysis (%): C=60.7; H=6.3; N=3.60; O=25.3; S=4.2
 2. u.v. and visible absorption bands:

    ______________________________________                                                 λ.sub.max (mμ)                                                                  E.sub.1 cm..sup.1%                                          ______________________________________                                                406         178                                                                350         (shoulder)                                                         297         305                                                                257         405                                                                224         518                                                         ______________________________________                                    


3. The most significant infrared absorption peaks in Nujol at the following frequencies (cm.⁻ ¹) (FIG. 4): 3700-3300 (s, br); 3300-3080 (m br); 3040-2780 (vs); 1460 (s); 1378 (s): Nujol; 1740 (m); 1700 (m); 1650 (s, br); 1605 (s, br); 1555 (s); 1510 (m, br); 1315 (w); 1275 (m); 1240 (m); 1220 (m); 1160 (m); 1090 (m); 1050 (m, br); 1020 (w); 970 (m); 945 (w); 910 (m); 808 (m); 765 (w); 720 (w),
 4. A complete figure of N.M.R. spectrum at 60 MH_(z) in CDCl₃ as in FIG. 7,
 5. rf value of 0.32 on silicagel plate using chloroform:methanol (95:5) as a solvent system.
 3. A rifamycin compound defined as rifamycin U having the following characteristics:a. U.V. and visible absorption bands:

    ______________________________________                                                 λ.sub. max (mμ)                                                                 E.sub.1 cm.sup.1%                                           ______________________________________                                                412         163                                                                353         (shoulder)                                                         299         265                                                                258         389                                                                220         451;                                                        ______________________________________                                    

B. the most significant absorption peaks in Nujol occur at the following frequencies (cm⁻ ¹): 3700-3100 (m, br); 3060 (vw); 3040-2740 (vs), 2720 (vs), 1460 (s), 1378 (m): Nujol; 1750-1705 (m, br); 1680-1620 (m, br); 1600 (s); 1558 (s); 1490 (w, shoulder); 1308 (w); 1270 (w, shoulder); 1245 (m, shoulder); 1225 (m); 1160 (m); 1120 (w); 1090 (vw); 1070 (w); 1030 (vw); 1020 (vw); 1000 (vw); 975 (w); 960 (w, shoulder); 905 (m); 850 (m); 802 (w); 800 (vw, shoulder); 777 (vw); 760 (vw); 720 (vw); 685 (vw); 670 (vw); C. an Rf value of 0.63 on silica gel plate using chloroform:methanol (95:5) as the solvent system;prepared by fermenting under aerobic conditions in the presence of an assimilable carbon source, an assimilable nitrogen source and essential mineral salts a mutant Nocardia mediterranea strain selected from the group consisting of Nocardia mediterranea ATCC 31064, Nocardia mediterranea ATCC 31065 and Nocardia mediterranea ATCC 31066 until the fermentation medium shows substantial antibiotic activity, filtering the fermentation medium, extracting the filtrate having a pH of about 7.5 to dissolve the product rifamycins P, Q, R and U and separating rifamycin U from the resulting solution by column chromatography. 